Process for preparing mechanical pulp by treating the pulp with an enzyme having cellobiohydralase activity

ABSTRACT

An enzymatic process for treating coarse pulp with an enzyme having cellobiohydrolase activity to reduce the specific energy requirements of the pulp and improve the properties of the pulp. Cellobiohydrolase enzymes isolated from the species Trichoderma, Aspergillus, Phanerochaete, Penicillium, Streptomyces, Humicola or Bacillus can be used.

The present invention relates to a process in accordance with thepreamble of claim 1 for preparing mechanical pulp.

According to a process of this kind, the wood raw material isdisintegrated into chips, which then are defibered to the desireddrainability, the raw material being subjected to an enzymatic treatmentduring the production process.

The invention also relates to an enzyme preparation according to thepreamble of claim 15, suitable for the treatment of mechanical pulp.

Chemical and mechanical pulps possess different chemical and fibretechnical properties and thus their use in different paper grades can bechosen according to these properties. Many paper grades contain bothtypes of pulps in different proportions according to the desiredproperties of the final paper products. Mechanical pulp is often used toimprove or to increase the stiffness, bulkiness or optical properties ofthe product.

In paper manufacture the raw material has first to be defibered.Mechanical pulp is mainly manufactured by grinding and refining methods,in which the raw material is subjected to periodical pressure impulses.Due to the friction heat, the structure of the wood is softened and itsstructure loosened, leading finally to separation of the fibres.

However, only a small part of the energy spent in the process is used toseparate the fibres, the major part being transformed to heat.Therefore, the total energy economy of these processes is very poor.

Several methods for improving the energy economy of mechanical pulpingare suggested in the prior art. Some of these are based on pretreatmentof chips by, e.g., water or acid (FI Patent Specifications Nos. 74493and 87371). Also known are methods which comprise treating the rawmaterial with enzymes to reduce the consumption of the refining energy.Thus, Finnish Patent Application No. 895676 describes an experiment inwhich once-refined pulp was treated with a xylanase enzyme preparation.It is stated in the application that this enzyme treatment would, tosome extent, decrease the energy consumption. In said prior artpublication the possibility of using cellulases is also mentioned, butno examples of these are given nor are their effects shown. As far asisolated, specified enzymes are concerned, in addition tohemicellulases, the interest has been focused on lignin modifyingenzymes, such as laccase. A treatment using the laccase enzyme did not,however, lead to decreased energy consumption.

In addition to the afore-mentioned isolated enzymes, the application ofgrowing white rot fungi in the manufacture of mechanical pulps has alsobeen studied. Carried out before defiberization, such a treatment with awhite rot fungus has been found to decrease the energy consumption andto improve the strength properties of these pulps. The drawbacks ofthese treatments are, however, the long treatment time needed (mostlyweeks), the decreased yield (85 to 95%), the difficulty to control theprocess and the impaired optical properties.

The aim of this method of invention is to remove the drawbacks of theknown techniques and to provide a completely new method for theproduction of mechanical pulp.

It is known that the amount and temperature of water bound to wood areof great importance for the energy consumption and quality of the pulp.The water bound to wood is known to decrease the softening temperatureof hemicelluloses and lignin between the fibres and simultaneously toweaken the interfibre bonding, which improves the separation of fibresfrom each other. During refining the energy is absorbed (bound) mainlyby the amorphous parts of the fibre material, i.e. the hemicellulose andlignin. Therefore, an increase of the portion of amorphous material inthe raw material improves the energy economy of the refining processes.

The invention is based on the concept of increasing the amorphousness ofthe raw material during mechanical pulping by treating the raw materialwith a suitable enzyme preparation, which reacts with the crystalline,insoluble cellulose.

The enzymes responsible for the modification and degradation ofcellulose are generally called "cellulases". These enzymes are comprisedof endo-β-glucanases, cellobiohydrolases and β-glucosidase. In simpleterms, even mixtures of these enzymes are often referred to as"cellulase", using the singular form. Very many organisms, such as woodrotting fungi, mold and bacteria are able to produce some or all ofthese enzymes. Depending on the type of organism and cultivationconditions, these enzymes are produced usually extracellularly indifferent ratios and amounts.

It is generally well known that cellulases, especiallycellobiohydrolases and endoglucanases, act strongly synergistically,i.e. the concerted, simultaneous effect of these enzymes is moreefficient than the sum of the effects of the individual enzymes usedalone. Such concerted action of enzymes, the synergism, is however,usually not desirable in the industrial applications of cellulases oncellulosic fibres. Therefore, it is often desired to exclude thecellulase enzymes totally or at least to decrease their amount. In someapplications very low amounts of cellulases are used for, e.g., removingthe fines, but in these applications the most soluble compounds arehydrolyzed to sugars in a limited hydrolysis as a result of the combinedaction of the enzymes.

In our experiments we have been able to show that a synergisticallyacting cellulase enzyme product, i.e. the "cellulase" cannot be used toimprove the manufacture of mechanical pulps because the application ofthis kind of enzyme product leads to the hydrolysis of insolublecellulose and thus impairs the strength properties of the fibres. Inconnection with the present invention, however, it has surprisingly beenfound that by using a cellulase enzyme preparation, which does notpossess a synergistic mode of action, cellulose can be modified in anadvantageous way and desired modifications can be achieved withoutremarkable hydrolysis or yield losses. Therefore, according to themethod of invention a cellulase preparation is used which exhibits asubstantial cellobiohydrolase activity and--compared with thecellobiohydrolase activity--a low endo-β-glucanase activity, if any.

Most cellulases are composed of functionally two different domains: thecore and the cellulose binding domain (CBD), in addition to the linkerregion combining these two domains. The active site of the enzyme issituated in the core. The function of the CBD is thought to be mainlyresponsible for the binding of the enzyme to the insoluble substrate. Ifthe tail is removed, the affinity and the activity of the enzyme towardshigh molecular weight and crystalline substrates is essentiallydecreased.

According to the process of the invention, the raw material to berefined is treated with an enzyme, able specifically to decrease thecrystallinity of cellulose. This enzyme can be e.g. cellobiohydrolase ora functional part of this enzyme and, as a cellulase enzyme preparation,it acts non-synergistically, as described above. In this context,"functional parts" designate primarily the core or the tail of theenzyme. Also mixtures of the above mentioned enzymes, obtainable by e.g.digestion (i.e. hydrolysis) of the native enzymes can be used.Comparable cellobiohydrolases are also produced by bacteria belonging tothe genus of Cellulomonas. The amorphous part of the raw material canalso be increased by certain polymerases (e.g. some endoglucanases).

Previously, no method has been presented, wherein only one (or several)biochemically characterized enzyme would have been used as the mainactivity to achieve a desired modification of the raw material. Theprior art contains methods and processes, in which the hydrolyticproperties of cellulases are exploited to produce sugars from differentcellulosic materials. In these applications, however, the aim is--incontrast to the process of the present invention,--to achieve the mostefficient synergistic action of the enzymes.

As used in the present application the term "enzyme preparation" refersto any such product, which contains at least one enzyme or a functionalpart of an enzyme. Thus, the enzyme preparation may be a culturefiltrate containing one or more enzymes, an isolated enzyme or a mixtureof two or several enzymes. "Cellulase" or "cellulase enzymepreparation", on the other hand, refers to an enzyme preparationcontaining at least one of the before mentioned cellulase enzymes.

For the purpose of the present application, the term "cellobiohydrolaseactivity" denotes an enzyme preparation, which is capable of modifyingthe crystalline parts of cellulose. Thus, the term "cellobiohydrolaseactivity" includes particularly those enzymes, which produce cellobiosefrom insoluble cellulose substrates. This term covers, however, also allenzymes, which do not have a clearly hydrolyzing effect or which onlypartially have this effect but which, in spite of this, modify thecrystalline structure of cellulose in such a way that the ratio of thecrystalline and amorphous parts of the lignocellulosic material isdiminished, i.e. the part of amorphous cellulose is increased. Theselast-mentioned enzymes are exemplified by the functional parts of e.g.cellobiohydrolase together or alone.

According to the process of the present invention, the enzyme treatmentis preferably carried out on the "coarse pulp" of a mechanical refiningprocess. This term refers in this application to a lignocellulosicmaterial, used as raw material of the mechanical pulp and which alreadyhas been subjected to some kind of fiberizing operation duringmechanical pulping e.g. by refining or grinding. Typically, thedrainability of the material to be enzymatically treated, is about 30 to1,000 ml, preferably about 100 to 700 ml. When applied directly to thechips, the enzyme treatment is usually not as efficient, because it isdifficult to achieve an efficient diffusion (adsorption) of the enzymepreparation into the fibres of the raw material, if still in the form ofchips. In contrast, e.g. a pulp, once refined, is well suited for use inthe method of invention. The term coarse pulp thus encompasses, e.g.,once refined or ground pulp, the rejects and long fibre fractions, andcombinations of these, which have been produced by thermomechanicalpulping (e.g. TMP) or by grinding (e.g. GW and PGW). It is essential forthe invention that the enzyme treatment be carried out at least beforethe final refining stage, where the material is refined to the desiredfreeness, which is typically less than 300 ml CSF, preferably less than100 ml CSF.

The process is not limited to a certain wood raw material, but it can beapplied generally to both soft and hard wood species, such as species ofthe order of Pinacae (e.g. the families of Picea and Pinus), Salicaceae(e.g. the family of Populus) and the species in the family of Betula.

According to the present invention the parts, in particular the core ofthe cellobiohydrolase enzyme can be used instead of thecellobiohydrolase for the manufacture of mechanical pulps. It has,namely, been observed that used in connection with the present process,that parts of the enzyme, in particular the core, have a similar,although weaker hydrolytic effect as the intact enzyme. Also the tail ofthe cellobiohydrolase enzyme has been observed to modify cellulose andis therefore suitable for the present invention.

According to a preferred embodiment the once-refined mechanical pulps ofCSF values of 30 to 1,000 ml are treated with the cellobiohydrolaseenzyme preparation at 30 to 90° C., in particular at 40 to 60° C., at aconsistency of 0.1 to 20%, preferably 1 to 10%. The treatment time is 1min to 20 h, preferably about 10 min to 10 h, in particular about 30 minto 5 h. The pH of the treatment is held neutral or slightly acid oralkaline, a typical pH being 3 to 10, preferably about 4 to 8. Theenzyme dosage varies according to the type of pulp and thecellobiohydrolase activity of the preparation, but is typically about 1μg to 100 mg of protein per gram of od. pulp. Preferably, the enzymedosage is about 10 μg to 10 mg of protein per gram of pulp.

The process according to the present invention can be combined withtreatments carried out with other enzymes, such as hemicellulases (e.g.xylanases, glucuronidases and mannanases) or esterases. In addition tothese enzymes, additional enzyme preparations containing β-glucosidaseactivity can be used in the present process, because this kind ofβ-glucosidase activity prevents the end product inhibition and increasesthe efficiency of the method.

Cellobiohydrolase enzyme preparations are produced by growing suitablemicro-organism strains, known to produce cellulase. The productionstrains can be bacteria, fungi or mold. As examples, the micro-organismsbelonging to the following species can be mentioned:

Trichoderma (e.g. T. reesei), Aspergillus (e.g. A. niger), Fusarium,Phanerochaete (e.g. P. chrysosporium; [Covert, S., Vanden Wymelenberg,A. & Cullen, D., Structure, organisation and transcription of acellobiohydrolase gene cluster from Phanerochaete chrysosporium, Appl.Environ. Microbiol. 58 (1992), 2168-2175], Penicillium (e.g. P.janthinellum, P. digitatum), Streptomyces (e.g. S. olivochromogenes, S.flavogriseus), Humicola (e.g. H. insolens), Cellulomonas (e.g. C. fimi)and Bacillus (e.g. B. subtilis, B. circulans, [Ito, S., Shikata, S.,Ozaki, K., Kawai, S., Okamoto, K., Inoue, S., Takei, A., Ohta, Y. &Satoh, T., Alkaline cellulase for laundry detergents: production byBacillus sp. KSM-635 and enzymatic properties, Agril. Biol. Chem. 53(1989), 1275-1281]. Also other fungi can be used, strains belonging tospecies, such as Phlebia, Ceriporiopsis and Trametes.

It is also possible to produce cellobiohydrolases or their functionalparts with strains, which have been genetically improved to producespecifically these proteins or by other genetically modified productionstrains, to which genes, coding these proteins, have been transferred.When the genes coding the desired protein(s) (Teeri, T., Salovuori, I. &Knowles, J., The molecular cloning of the major cellobiohydrolase genefrom Trichoderma reesei Bio/Technology 1 (1983), 696-699) have beencloned it is possible to produce the protein or its part in the desiredhost organism. The desired host may be the fungus T. reesei (Mitsuishi,Y., Nitisinprasert, S., Saloheimo, M., Biese, I., Reinikainen, T.,Clayssens, M., Keranen, S., Knowles, J. & Teeri, T., Site-directedmutagenesis of the putative catalysic residues of Trichoderma reeseicellobiohydrolase I and endoglucanase I, FEBS Lett. 275 (1990),135-138), a yeast (Penttila, M., Antre, L., Lehtovaara, P., Bailey, M.,Teeri, T. & Knowles, J., Efficient secretion of two fungalcellobiohydrolases by Saccharomyces cerevisiae. Gene 63 (1988) 103-112)or some other fungus or mold, from species such as Aspergillus (19), abacterium or any other micro-organism, whose genome is sufficientlyknown.

According to a preferred embodiment the desired cellobiohydrolase isproduced by the fungus Trichoderma reesei. This strain is a generallyused production organism and its cellulases are fairly well known. T.reesei synthesizes two cellobiohydrolases, which are later referred toas CBH I and CBH II, several endoglucanases and at least twoβ-glucosidases (Chen, H., Hayn, M. & Esterbauer, H., Purification andcharacterization of two extracellular β-glucosidases from Trichodermareesei, Biochim. Biophys. Acta 1121 (1992) 54-60). The biochemicalproperties of these enzymes have been extensively described on purecellulosic substrates. Endoglucanases are typically active on solubleand amorphous substrates (CMC, HEC, β-glucan), whereas thecellobiohydrolases are able to hydrolyze only crystalline cellulose. Thecellobiohydrolases act clearly synergistically on crystallinesubstrates, but their hydrolysis mechanisms are supposed to be differentfrom each other. The present knowledge on the hydrolysis mechanism ofcellulases is based on results obtained on pure cellulose substrates,and may not be valid in cases, where the substrate contains also othercomponents, such as lignin or hemicellulose.

The cellulases of T. reesei (cellobiohydrolases and endoglucanases) donot essentially differ from each other with respect to their optimalexternal conditions, such as pH or temperature. Instead they differ fromeach other with respect to their ability to hydrolyze and modifycellulose in the wood raw material.

As far as their enzymatic activities are concerned, thecellobiohydrolases I and II differ also to some extent from each other.These properties can be exploited in the present invention. Therefore,it is particularly preferable to use cellobiohydrolase I (CBH I)produced by T. reesei according to the present invention for reducingthe specific energy consumption of mechanical pulps. The pI value ofthis enzyme is, according to data presented in the literature, 3.2 to4.2 depending on the form of the isoenzyme (20) or 4.0 to 4.4, whendetermined according to the method presented in Example 2. The molecularweight is about 64,000 when determined by SDS-PAGE. It must be observed,however, that there is always an inaccuracy of about 10% in the SDS-PAGEmethod. Cellobiohydrolases alone or combined to e.g. hemicellulases canbe particularly preferably used for the modification of the propertiesof mechanical pulps, e.g. for improving the technical properties of thepaper (i.e. the handsheet properties) prepared from these pulps.Naturally, also mixtures of cellobiohydrolases can be used for thetreatment of pulps, as described in Example 6.

Cellobiohydrolase can be separated from the culture filtrates of thefungus Trichoderma reesei by several conventional, known methods.Typically, in these separation and isolation methods several differentpurification techniques, such as precipitation, ion exchangechromatography, affinity chromatography and gel permeationchromatography can be used and combined. By using affinitychromatography, cellobiohydrolase can be separated easily even directlyfrom the culture filtrate (van Tilbeurgh, H. Bhikhabhai, R. Pettersson,L. and Claeyessens M. (1984), Separation of endo- and exo-typecellulases using a new affinity method. FEBS Lett. 169, 215-218). Thepreparation of the gel material needed for this affinity chromatographyis, however, difficult and this material is not commercially available.According to a preferred embodiment of the invention, thecellobiohydrolase I enzyme is separated from the other proteins of theculture filtrate by a rapid purification method, based on anionexchange. This method is described in detail in Example 1. The method ofinvention is not, however, limited to this isolation method of proteins,but it is also possible to isolate or enrich the desired protein byother known methods.

Significant advantages can be obtained with this invention. Thus, withthis method the specific energy consumption can be remarkably decreased;as the examples described below show, an energy saving of up to 20% canbe achieved using the method of invention, as compared with untreatedraw materials. Using a suitable cellobiohydrolase, also the propertiesof the pulp can be improved. According to the method of invention, inwhich the synergistic action of the enzyme preparation used is absent oronly insignificant, also the problems involved in the above mentionedfungal treatments can be avoided. Thus, the treatment time lasts onlyfor few hours, the yield is extremely high, the quality of the pulp isgood and the connection of the method to the present processes issimple.

The method can be applied in all mechanical or semimechanical pulpingmethods, such as in the manufacture of ground wood (GW, PGW),thermomechanical pulps (TMP) and chemimechanical pulps (CTMP).

In the following the invention will be examined in more detail with theaid of the following non-limiting examples.

EXAMPLE 1 Purification of Cellobiohydrolase I

The fungus Trichoderma reesei (strain VTT-D-86271, RUT C-30) was grownin a 2 m³ fermenter on a media containing 3% (w/w) Solka floc cellulose,3% corn steep liquor, 1.5% KH₂ PO₄ and 0.5% (NH₄)₂ SO₄. The temperaturewas 29° C. and the pH was controlled between 3.3 and 5.3. The culturetime was 5 d, whereafter the fungal mycelium was separated by a drumfilter and the culture filtrate was treated with bentonite, as describedby Zurbriggen et al. (Zurbriggen, B. Z., Bailey, M. J., Penttila, M. E.,Poutanen, K. and Linko M. (1990), Pilot scale production of heterologousTrichoderma reesei cellulase in Saccharomyces cerevisiae. J. Biotechnol.13, 267-278). After this the liquor was concentrated by ultrafiltration.

The isolation of the enzyme was started by buffering the concentrate bygel filtration to pH 7.2 (Sephadex G-25 coarse). The enzyme solution wasapplied at this pH (7.2) to an anion exchange chromatography column(DEAE-Sepharose FF), to which most of the proteins in the sample,including CBH I, were bound. Most of the proteins bound to the columnincluding also other cellulases than CBH I were eluated with a buffer(pH 7.2) to which sodium chloride was added to form a gradient in theeluent buffer from 0 to 0.12 M. The column was washed with a buffer atpH 7.2, containing 0.12 M NaCl, until no significant amount of proteinwas eluted. CBH I was eluted by increasing the concentration of NaCl to0.15 M. The purified CBH I was collected from fractions eluted by thisbuffer.

EXAMPLE 2 Characterization of CBH I

The protein properties of the enzyme preparation purified according toexample 1 were determined according to usual methods of proteinchemistry. The isoelectric focusing was run using a Pharmacia MultiphorII System apparatus according to the manufacturer's instructions using a5% polyacrylamide gel. The pH gradient was achieved by using a carrierampholyte Ampholine, pH 3.5-10 (Pharmacia), where a pH gradient between3.5 and 10 in the isoelectric focusing was formed. A conventional gelelectrophoresis under denaturating conditions (SDS-PAGE) was carried outaccording to Laemmli, (Laemmli, U. K., Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4. Nature 227 (1970),680-685; Chen H., Hayn M. & Esterbauer H. Purification andcharacterization of two extracellular β-glucosidases from Trichodermareesei. Biochim. Biophys. Acta 1121 (1992), 54-60), using a 10%polyacrylamide gel. In both gels the proteins were stained with silverstaining (Bio Rad, Silver Stain Kit).

For CBH I the molecular weight obtained was 64,000 and the isoelectricpoint 4.0-4.4. As judged from the gels, over 90% of the proteinsconsisted of CBH I.

EXAMPLE 3 Enzymatic Treatment

The ability of the enzyme produced and characterized according to theexamples 1 and 2 to hydrolyze coarse wood fibres (spruce) were studiedand compared with other cellulases. The enzyme dosage was 0.5 mg/g ofpulp and the hydrolysis conditions were: pH 5-5.5. temperature 45° C.,hydrolysis time 24 h. The results are described in Table 1. It isnoteworthy that cellobiohydrolases alone did not achieve substantialformation of sugars and thus not yield losses.

                  TABLE 1                                                         ______________________________________                                        Hydrolysis of coarse pulp (spruce) with different cellulases                                        Degree of hydrolysis, %                                 Enzyme     Reducing sugars,g/l                                                                        of d.w.                                               ______________________________________                                        CBH I    0.003        0.01                                                    CBH II     0.05                        0.1                                    EG I         0.06                      0.12                                   EG II       0.04                       0.08                                   ______________________________________                                    

EXAMPLE 4 Effect of Enzymatic Treatment on the Swelling of Fibres

The long fibre fraction (+48) of the fractionated TMP spruce pulp wastreated with cellulases at 5% consistency at 45° C. for 24 hours. Thepulp was suspended in tap water and pH was adjusted between 5-5.5 usingdiluted sulphuric acid. The enzyme dosage was 0.5 mg/g of dry pulp.After the treatment the pulp was washed with water and the WRV (waterretention value) describing the swelling of the fibres was determined bya SCAN method. The results are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Swelling of spruce fibres after the enzymatic treatment                              Enzyme                                                                              WRV, %                                                           ______________________________________                                               CBH I 108                                                                     Control                                                                              102                                                             ______________________________________                                    

According to the results CBH I is able to modify the pulp by increasingthe ability to adsorb water, which improves the refining.

EXAMPLE 5 Effect of Enzyme Treatment on the Flexibility of the Fibres

The long fibre fraction (+48) of the fractionated TMP spruce pulp wastreated with CBH I at 5% consistency at 45° C. for 2 hours. The enzymedosage was 1 mg CBH/g of dry pulp. After the treatment the flexibilityof the fibres was measured using a hydrodynamic method. From each samplethe flexibility of 100-200 individual fibres was measured. The resultsare presented in Table 3. According to the results the stiffness of thefibres was decreased; i.e. flexibility of the fibres was increased afterthe CBH treatment.

                  TABLE 3                                                         ______________________________________                                        The effect of the enzyme treatment on the flexibility (stiffness)             of the fibres                                                                 Flexibility index                                                             (10.sup.-12 Nm.sup.2)                                                                              Control                                                                                         CBH I                                  ______________________________________                                        Smallest value    2.7     2.1                                                 Lower quartile                             7.2                                Median                                    14.26.8                             Upper quartile                            21.8                                Greatest value                            40.2                                Mean                                      15.8 17.7                           Standard deviation                                                                                                      9.6                                 ______________________________________                                    

EXAMPLE 6 Effect of Enzymatic Treatment on the Specific EnergyConsumption of Refining

In three independent series, coarse once refined TMP pulps, withfreeness values (CSF) of 450-550 ml, were treated with CBH I enzymepreparation. The consistency of the pulp suspension in each experimentwas 5% in tap water, the treatment time 2 h and temperature 45-50° C.The amount of pulp treated was 1 kg of dry pulp and the enzyme dosage0.5 mg/g of pulp. After the enzyme treatment the pulps were drained,centrifuged and homogenized. The reference samples were treated in thesame way, but without enzyme addition.

The pulps were further refined using a Bauer or a Sprout Waldron singlerotating disk atmospheric refiner using decreasing plate settings. Therefining was followed by determining the freeness values of theintermediate samples and stopped when the freeness values were below 100ml. The energy consumption in each refining experiment was measured andthe specific energy consumption was calculated and reported as kWh/kgo.d. weight basis. The results are presented in Table 4.

                  TABLE 4                                                         ______________________________________                                        The specific energy consumption on untreated samples and the CBH I and        CBH I/CBH II treated samples in four independent test series.                 The values of the specific energy consumption are reported                    at the CSF level of 100 ml.                                                               Test 1  Test 2    Test 3                                                                              Test 4                                    Sample                  kWh/kg                                                                                   kWh/kg                                                                             kWh/kg                                ______________________________________                                        CBH I       1.73    1.64      2.04  1.81                                      CBH I digested                                                                               --         --              1.76                                CBH I/CBH II                                                                                   --       --              1.77                                Controls                  2.05                                                                                          2.08                                ______________________________________                                    

It can be observed from the results obtained that it is possible toreduce the energy consumption by using the CBH I enzyme by 15-20% ascompared with the reference sample. The same effect was also obtained,when the preparation contained both cellobiohydrolase activities or theproteolytically digested CBH. The latter enzyme preparation containedboth functional domains of CBH I i.e. the core and the CBD.

EXAMPLE 7 Effect of the Enzyme Treatment on Handsheet Properties of thePulps

Spruce TMP pulp was treated with an enzyme preparation containing CBH Iand CBH II and further refined. Improvment of the strength properties ofenzyme treated pulp can be observed as compared to the untreatedcontrol.

                  TABLE 5                                                         ______________________________________                                        Strength properties of the CBH I + CBH II treated                             sample and the untreated                                                      control at the CSF level of 150 ml                                                           Tensile index,                                                                            Tear index,                                        Sample                                mNm.sup.2 /kg                           ______________________________________                                        Control        31.3       7.0                                                 CBH I + CBH II     32.0               7.2                                     ______________________________________                                    

EXAMPLE 8 Effect of the Enzyme Treatment on the Crystallinity ofCellulose

Spruce TMP pulps were treated with the intact cellobiohydrolases andwith the digested CBHs. Decrease in the crystallinity of the pulp wasdetected. The same effect was not observed with endoglucanases (EG I andEG II).

We claim:
 1. A process for preparing mechanical pulp from woodraw-material, which comprises:(i) refining or grinding wood raw materialto obtain a coarse pulp having a drainability of from about 30 to 1,000CSF; (ii) treating the coarse pulp with an enzyme having acellobiohydrolase activity effective for modifying crystalline parts ofcellulose and as compared with the cellobiohydrolase activity, a lowendo-β-glucanase activity, wherein the endo-β-glucanase activity is lessthan that which will significantly hydrolyze the cellulose; and (iii)mechanically defibering the enzyme treated coarse pulp to lower thefreeness to a value of less than 300 ml CSF.
 2. A process according toclaim 1, wherein an enzyme preparation is used, which contains isolatedcellobiohydrolase enzymes or parts thereof.
 3. A process according toclaim 1, wherein an enzyme preparation is used, which has been producedby cultivating on a suitable growth medium a microorganism strainbelonging to the species Trichoderma, Aspergillus, Phanerochaete,Penicillium, Streptomyces, Humicola or Bacillus.
 4. A process accordingto claim 3, wherein the enzyme preparation used has been produced by astrain genetically improved for producing an enzyme havingcellobiohydrolase activity, or by a strain to which the gene coding forsaid activity has been transferred.
 5. A process according to claim 4,wherein the cellobiohydrolase enzyme used has been separated from theother proteins of the growth medium by a purification method based onrapid anionic ion exchange.
 6. A process according to claim 3, whereinthe cellobiohydrolase enzyme used has been separated from the otherproteins of the growth medium by a purification method based on rapidanionic ion exchange.
 7. A process according to claim 1, wherein theenzyme preparation used contains cellobiohydrolase produced by themicroorganism Trichoderma reesei.
 8. A process according to claim 7,wherein the cellobiohydrolase enzyme used has been separated from theother proteins of the growth medium by a purification method based onrapid anionic ion exchange.
 9. A process according to claim 7, whereinthe enzyme preparation used contains the cellobiohydrolase I (CBH I)produced by the fungus strain Trichoderma reesei having a molecularweight, determined by SDS-PAGE, of about 64,000 and an isoelektric pointof about 3.2 to 4.4.
 10. A process according to claim 1, wherein theenzyme treatment is carried out at 30 to 90° C. and at a consistency ofabout 0.1-20%, the duration of the treatment being about 1 min-20 h. 11.A process according to claim 10, wherein the enzyme treatment is carriedout at about 40 to 60° C.
 12. A process according to claim 11, whereinthe enzyme treatment is carried out at a consistency of about 0.1-10%.13. A process according to claim 12, wherein the duration of thetreatment is about 30 min-5 h.
 14. A process according to claim 11,wherein the duration of the treatment is about 30 min-5 h.
 15. A processaccording to claim 10, wherein the enzyme treatment is carried out at aconsistency of about 0.1-10%.
 16. A process according to claim 10,wherein the duration of the treatment is about 30 min-5 h.
 17. A processaccording to claim 1, wherein the enzyme preparation is dosaged in anamount of about 10 μg-100 mg protein per gram of dry pulp.
 18. A processaccording to claim 17, which comprises enzymatically treating coarsepulp having a drainability of about 300 to 700 ml CSF.
 19. A processaccording to claim 17, wherein the enzyme preparation is dosaged in anamount of about 100 μg-10 mg protein per gram of dry pulp.
 20. A processaccording to claim 1, wherein the mechanical pulp is prepared by the GW,PGW, TMP or CTMP process.
 21. A process for preparing mechanical pulpfrom wood raw-material, which comprises:(i) refining or grinding woodraw material to obtain a coarse pulp having a drainability of from about30 to 1,000 CSF; and (ii) increasing the amorphous portion of saidcoarse pulp by treating the coarse pulp with an enzyme having acellobiohydrolase activity effective for modifying crystalline parts ofcellulose and as compared with the cellobiohydrolase activity, a lowendo-β-glucanase activity, wherein the endo-β-glucanase activity is lessthan that which will significantly hydrolyze the cellulose; and (iii)mechanically defibering the enzyme treated coarse pulp to lower thefreeness to a value of less that 300 ml CSF.
 22. A process according toclaim 21, wherein an enzyme preparation is used, which contains isolatedcellobiohydrolase enzymes or parts thereof.